An overview of <i>Phoneutria nigriventer</i> spider venom using combined transcriptomic and proteomic approaches

Phoneutria nigriventer is one of the largest existing true spiders and one of the few considered medically relevant. Its venom contains several neurotoxic peptides that act on different ion channels and chemical receptors of vertebrates and invertebrates. Some of these venom toxins have been shown as promising models for pharmaceutical or biotechnological use. However, the large diversity and the predominance of low molecular weight toxins in this venom have hampered the identification and deep investigation of the less abundant toxins and the proteins with high molecular weight. Here, we combined conventional and next-generation cDNA sequencing with Multidimensional Protein Identification Technology (MudPIT), to obtain an in-depth panorama of the composition of P. nigriventer spider venom. The results from these three approaches showed that cysteine-rich peptide toxins are the most abundant components in this venom and most of them contain the Inhibitor Cysteine Knot (ICK) structural motif. Ninety-eight sequences corresponding to cysteine-rich peptide toxins were identified by the three methodologies and many of them were considered as putative novel toxins, due to the low similarity to previously described toxins. Furthermore, using next-generation sequencing we identified families of several other classes of toxins, including CAPs (Cysteine Rich Secretory Protein—CRiSP, antigen 5 and Pathogenesis-Related 1—PR-1), serine proteinases, TCTPs (translationally controlled tumor proteins), proteinase inhibitors, metalloproteinases and hyaluronidases, which have been poorly described for this venom. This study provides an overview of the molecular diversity of P. nigriventer venom, revealing several novel components and providing a better basis to understand its toxicity and pharmacological activities.巴西游走蛛（Phoneutria nigriventer）是现存体型最大的真蜘蛛类群之一，亦是少数具备医学相关性的蜘蛛物种。其毒液含有多种神经毒性肽，可靶向作用于脊椎动物与无脊椎动物的不同离子通道及化学受体。其中部分毒液毒素已被证实为极具潜力的医药或生物技术应用模型。然而，该毒液中毒素的高度多样性，以及低分子量毒素占主导的组成特征，极大阻碍了对丰度较低的毒素与高分子量蛋白质的鉴定及深入研究。本研究结合传统cDNA测序、新一代cDNA测序与多维蛋白质鉴定技术（Multidimensional Protein Identification Technology，MudPIT），对巴西游走蛛毒液的组成进行了全景式深入解析。三种技术的结果显示，富含半胱氨酸的肽类毒素是该毒液中丰度最高的组分，且其中绝大多数含有胱氨酸结抑制基序（Inhibitor Cysteine Knot，ICK）。通过这三种方法共鉴定出98条对应富含半胱氨酸肽类毒素的序列，由于多数序列与已报道的毒素相似性极低，因此被认定为推定新型毒素。此外，借助新一代测序技术，本研究还鉴定出多个其他类别的毒素家族，包括CAP家族（富含半胱氨酸分泌蛋白（Cysteine Rich Secretory Protein，CRiSP）、抗原5及病程相关蛋白1（Pathogenesis-Related 1，PR-1））、丝氨酸蛋白酶、翻译控制肿瘤蛋白（translationally controlled tumor proteins，TCTPs）、蛋白酶抑制剂、金属蛋白酶及透明质酸酶——这类毒素在该毒液中的相关研究此前较为匮乏。本研究全面梳理了巴西游走蛛毒液的分子多样性，揭示了多种新型组分，为理解其毒性与药理活性提供了更为坚实的理论基础。